Many modern treatments for human diseases employ or target proteins. To develop such treatments, physicians and scientists require knowledge of the amino acid sequences, structures, and abundances of proteins that are expressed in the human body in various contexts, as well as interactions among these proteins. A key aspect of studying and manipulating proteins is detection. Proteins can be detected in vivo or in vitro, for purposes of analyzing or preparing protein samples, using a variety of techniques. Many of these techniques involve contacting proteins with a detectable binding partner.
Detection of proteins in electrophoresis gels is frequently performed using colored or fluorescent protein stains such as Coomassie Brilliant Blue or SYPRO Ruby. These stains can bind to proteins non-covalently, in a manner that is largely independent of amino acid sequence, and can be visualized upon illumination with specific wavelengths of light. Protein stains allow robust and sensitive detection, but hinder the rapid processing of biological samples for downstream applications. The process of applying the stain to the gel (staining) prior to detection can take hours. Similarly time consuming is the removal of the stain after detection (destaining), which can be necessary in order to obtain purified proteins when the gel is used preparatively. Staining and destaining involve agitation of the gel, prolonged immersion of the gel in an aqueous buffer, and frequent changes of this buffer. During these processes, some proteins (particularly low-molecular-weight and hydrophilic proteins) can diffuse out of the gel and into the buffer, thereby becoming lost to subsequent analysis. Use of protein stains, and the accompanying gel handling, can therefore reduce the number of proteins that can be identified after electrophoresis.
Alternatively, proteins can be made detectable by contacting the proteins with a halo-substituted organic compound and exposing the proteins to UV light. As disclosed by Edwards et al. in U.S. Pat. No. 7,569,103 B2 (Aug. 4, 2009) and U.S. Pat. No. 8,007,646 B2 (Aug. 30, 2011), and elsewhere, this procedure causes a UV-induced reaction between the indole moiety of tryptophan and the halo-substituted organic compound. Reacted tryptophan residues are covalently modified and fluorescent, undergoing excitation at the same wavelengths used to induce the reaction and emitting in the visible range. The reaction, and the associated reagents, apparatus, and methods used to perform the reaction and detect products thereof, are sometimes referred to by the name ‘Stain-Free™’ (Bio-Rad).
Proteins can be contacted with a halo-substituted organic compound and reacted before or after gel electrophoresis. Contact can also occur during electrophoresis in situ if the halo-substituted organic compound is a constituent of the gel. Detection of proteins using covalently modified tryptophan fluorescence, compared with using protein stains, is often more convenient and economical, and is also not limited to applications involving electrophoresis gels. For example, proteins can be contacted with the halo-substituted organic compound, reacted, and detected while suspended in solution or deposited on a blotting membrane.
Tryptophan and other aromatic amino acid side-chains can be protected from covalent modification by binding non-covalently to cyclodextrins, which are reviewed by Del Valle (E. M. M. Del Valle, Process Biochemistry 39, 1033-1046, 2004) and Serno et al. (T. Serno et al., Advanced Drug Delivery Reviews 63, 1086-1106, 2011). Cyclodextrins are cyclic oligosaccharides that include several (typically 6, 7, or 8) glucopyranose units joined by α-(1,4) bonds. In a cyclodextrin molecule, the glucopyranose units form a tapered annular structure with a hydrophobic cavity at the center. Upon contact with a cyclodextrin, an aromatic amino acid side-chain can be inserted into the cavity, thereby becoming sequestered from further chemical interactions. Cyclodextrins have been used for example as excipients in protein-based drugs, and can prevent protein aggregation by limiting protein-protein interactions involving aromatic amino acids.
In addition to the standard α-, β-, and γ-cyclodextrins, varieties of cyclodextrins with substituted glucopyranose units have been characterized. The substituents, in most cases attached at the 2, 3, and 6 positions, include alkyl, hydroxyalkyl, and carboxyalkyl groups. Cyclodextrins covalently linked to one or more amino acids have also been synthesized (see, for example, Djedaïni-Pilard et al., J. Chem. Soc. Perkin Trans. 2, 723-730, 1995).
Cyclodextrins are one class of cyclic organic molecules that can non-covalently bind smaller molecules to form supramolecular complexes. Such a cyclic molecule serves as the ‘host’ in host-guest chemistry (reviewed, for example, by H.-J. Schneider and A. K. Yatsimirsky in Chem. Soc. Rev., 37, 263-277, 2008), and contains a central cavity to accommodate one or more appropriately sized ‘guest’ molecules through hydrogen bonding, ionic bonding, Van der Waals interactions, and/or hydrophobic interactions. The cyclic molecule can be an oligomer or polymer of repeating units, and can be referred to as a macrocycle. Other examples of cyclic organic host molecules include cucurbiturils, which are comprised of glycoluril monomers linked by methylene bridges. Cucurbit[8]uril has been shown to bind methyl viologen with 1:1 stoichiometry, and simultaneously accommodate methyl viologen and an amino acid in its cavity. Further, cucurbit[8]uril demonstrates binding specificity for the aromatic amino acids tryptophan, tyrosine, and phenylalanine (see P. Rajgariah and A. R. Urbach, J. Incl. Phenom. Macrocycl. Chem. 62, 251-254, 2008). Still other cyclic organic host molecules include pillararenes, calixarenes, and crown ethers.